Relevance: Parasitic protozoa such as Leishmania and Trypanosoma infect millions of people worldwide and cause devastating and fatal diseases. This application will elucidate an important mechanism whereby these parasites successfully invade human cells and cause disease. Summary. Recent discoveries have revealed that the whip-like flagellum that mediates motility of these single-cell parasites plays a critical role in he disease causing stages, the amastigote of Leishmania and the bloodstream form African trypanosomes. Specifically, our laboratory has discovered a novel protein, KHARON1, that is involved in targeting integral membrane proteins to the flagellar membrane in Leishmania mexicana. Importantly, KHARON1 is essential for disease-causing amastigotes of L. mexicana to survive inside the phagolysosomal vesicles of human macrophages. Although ?kharon1 null mutants replicate as axenic amastigotes in culture medium, once they enter host macrophages they do not replicate but rather die inside these host cells. These results suggest a role for KHARON1 specifically in survival of amastigotes inside the macrophage phagolysosome. Recent experiments also indicate that ?kharon1null mutants are avirulent following infection of Balb/C mice. Other preliminary data establish that KHARON1 is part of a high molecular weight multi-protein complex designated the KHARON Complex. The overall objective of this application is to dissect the critical function of the KHARON Complex in disease causing amastigotes and to thus illuminate the role of the amastigote flagellum in parasite virulence. The KHARON1 protein, which localizes to the base of the flagellar axoneme in insect stage promastigotes, will be localized in amastigotes by high-resolution fluorescence microscopy and electron microscopy. The ability of KHARON1 to mediate trafficking of a membrane protein to the amastigote flagellum will be demonstrated to confirm that this protein is involved in flagellar membrane targeting in amastigotes as well as promastigotes. Recently published experiments have demonstrated that the tips of the amastigote flagella form `synapses' with the phagolysomal membrane of the macrophage, suggesting that these synapses could be important in parasite/macrophage interactions. The possibility that ?kharon1 null mutants may fail to form such potentially critical synapses will be tested by electron microscopy. Additionally the possibility that ?kharon1 mutants fail to deliver a protein from the flagellum to the macrophage cytosol will also be examined. These experiments could provide a mechanistic explanation for the avirulence of ?kharon1 null mutants. Finally, preliminary studies have identified several candidate components of the KHARON Complex using biotin proximity labeling. Experiments will address whether these candidate subunits exist in the same high molecular weight complex as KHARON1 in intracellular amastigotes. Overall, this application will elucidate the critical role of the KHARON Complex in parasite virulence.